The production of recombinant glycoproteins has been an area of great activity for the biotechnology industry. One drawback of recombinant glycoproteins has been the heterogeneity of glycosylation produced by commonly used cellular host such as CHO cells. In contrast, the present invention provides lower eukaryotic host cells that have been engineered to produce recombinant erythropoietins comprising pre-selected desired N-glycan structures. The compositions of recombinant erythropoietins produced therefrom are significantly greater in uniformity of glycoforms than those produced in CHO cells.
Erythropoietin is a protein hormone which has been widely used for therapeutic indications requiring increased formation of red blood cells including anemia due to renal failure or chemotherapy treatment. Because of the great demand for safe and effective erythropoietin, recombinant human erythropoietin has become the largest selling recombinant human protein product.
Native human erythropoietin contains four carbohydrate chains (three N-linked and one O-linked). The protein requires tri- and tetra-antennary sialylated N-glycans for maximum in vivo efficacy. However, in vitro receptor binding and cell-based assays reveal that erythropoietins with multi-branched sialylated glycans and erythropoietins with additional N-glycosylation sites exhibit decreased binding relative to enzymatically deglycosylated erythropoietin. This paradox can be explained by considering clearance from the circulatory system. Tetra-antennary sialylated erythropoietin and darbepoetin exhibit longer serum half-lives compared with bi-antennary sialylated and nonglycosylated erythropoietin. (The principal routes of clearance for erythropoietin are via renal filtration, through binding to the asialoglycoprotein receptor, endothelial cell uptake and internalization by the target cell through the erythropoietin receptor.)
Currently marketed forms of recombinant erythropoietin include Epogen with three tetra-antennary N-glycan structures and Aranesp, erythropoietin engineered to contain two additional N-glycosylation sites for a total of five tetra-antennary N-glycan structures. The addition of these extra N-glycan attachment sites has resulted in a longer serum half-life and consequently an increased in vivo activity of the hormone. These erythropoietins are produced from CHO cells and secreted with a heterogeneous mixture of N-glycan structures. Process development is used to enrich for the tetra-antennary sialylated glycoform (see FIG. 1).
Past efforts to improve upon the properties of erythropoietin have included efforts to alter the glycosylation, for example by adding glycosylation sites, as well as efforts to conjugate the protein to polymers, such as polyethylene glycols. See, for example EP 0640619; WO 00/32772 WO 01/02017 and WO 03/029291. Despite these attempts, there remains a need for recombinant erythropoietin having improved properties such as greater ease of administration; less rigorous dosage regimens; improved pharmacokinetics and bioavailability; and less expensive manufacture. In particular, robust processes and materials useful to produce compositions of erythropoietin possessing these qualities from lower eukaryotic cells has remained an elusive and desirable goal.